REDOX FTIR DIFFERENCE SPECTROSCOPY USING CAGED ELECTRONS REVEALS CONTRIBUTIONS OF CARBOXYL GROUPS TO THE CATALYTIC MECHANISM OF HEME-COPPEROXIDASES

Redox spectra of the haem-copper oxidases cytochrome aa(3) of Rhodobac ter sphaeroides and cytochrome bo(3) of Escherichia coli were recorded in the visible and infrared spectral regions, The reduction of oxidas es was initiated after light activation of the 'caged electron' donor riboflavin, Infrared redox difference spectra exhibit absorbance chang es in the amide I region, which are indicative of very small redox-lin ked conformational movements in the polypeptide backbone, A reproducib le redox-dependent pattern of positive and negative absorption changes is found in the carbonyl region (1680-1750 cm(-1)). The carbonyl band s shift to lower frequencies due to cm isotope exchange of the solvent H2O to D2O. This common feature of cytochrome c and quinol oxidases i ndicates that at least (i) one redox-sensitive carboxyl group is in th e protonated state in the oxidized form and (ii) one carboxylic acid i s involved at a catalytic step - presumably in proton translocation - of haem-copper oxidases.